Hepatitis C virus (HCV) infections represent a worldwide health problem, with approximately 0.5 to 1.5% of most populations testing positive for antibodies to HCV. An estimated 50% of infected individuals will become chronic carriers of HCV and will be at increased risk for the development of cirrhosis and hepatocellular carcinoma and will also act as a source of infection for others. Transmission of HCV occurs most readily through contact with contaminated blood as in blood transfusions and IV drug abuse; however, the true epidemiology of this agent is only now emerging through the use of diagnostic assays. Other routes of transmission include sexual contact, intrafamilial contact, and transmission from mother to child at birth. The development of a vaccine for this virus must be considered a high priority. Currently, the only available method of testing vaccine candidates is the immunization and challenge of chimpanzees, a very costly and time consuming approach. The development of an in vitro assay for evaluating the presence of neutralizing antibodies would greatly facilitate vaccine development. This proposal will employ a recently developed primary hepatocyte tissue culture system for the in vitro propagation of HCV to address these needs. The specific aims of this proposal are: 1) to optimize methods for detecting HCV polypeptides using a human hepatoma cell line transfected with recombinant expression vectors encoding various domains of the HCV polyprotein; 2) to produce antibodies to the capsid and envelope proteins of HCV expressed in the insect cell/baculovirus system; 3) to define the authentic HCV proteins expressed in HCV-infected primary hepatocytes using immunoprecipitation and immunoblot methodology; 4) to develop a quantitative assay for measuring in vitro infections of primary hepatocytes with HCV and to use this assay to develop an in vitro neutralization assay for HCV; 5) to examine panels of human and chimpanzee sera for the presence of neutralizing antibodies and to evaluate antisera produced against the envelope proteins of HCV for neutralizing activity; and 6) to create immortalized human and chimpanzee hepatocyte cell lines permissive for HCV infections.